Phl p 5a derivatives having reduced allergeneity and retained T-cell reactivity

ABSTRACT

The present invention relates to the preparation and use of variants of the group 5 allergen of the Pooideae which are characterized by reduced IgE reactivity compared with the known wild-type allergens and at the same time by substantially retained reactivity with T lymphocytes. These hypoallergenic allergen variants can be employed for the specific immunotherapy (hyposensitization) of patients having grass pollen allergy or for the preventative immunotherapy of grass pollen allergies.

This application is a U.S. National Stage application under §371 of theInternational application PCT/EP04/04848, filed May 6, 2004, which isincorporated by reference herein in its entirety.

The present invention relates to the preparation and use of variants ofthe group 5 allergen of the Pooideae which are characterised by reducedIgE reactivity compared with the known wild-type allergens and at thesame time by substantially retained reactivity with T lymphocytes.

These hypoallergenic allergen variants can be employed for the specificimmunotherapy (hyposensitisation) of patients having grass pollenallergy or for the preventative immunotherapy of grass pollen allergies.

A preferred embodiment of the invention relates to variants of the majorallergen Phl p 5a from the pollen of timothy grass (Phleum pratense).

BACKGROUND OF THE INVENTION

Type 1 allergies are of importance worldwide. Up to 20% of thepopulation in industrialised countries suffer from complaints such asallergic rhinitis, conjunctivitis or bronchial asthma. These allergiesare caused by allergens present in the air (aeroallergens) which areliberated from sources of various origin, such as plant pollen, mites,cats or dogs. Up to 40% of these type 1 allergy sufferers in turnexhibit specific IgE reactivity with grass pollen allergens (Freidhoffet al., 1986, J. Allergy Clin. Immunol. 78, 1190-2002).

The substances which trigger type 1 allergy are proteins, glycoproteinsor polypeptides. After uptake via the mucous membranes, these allergensreact with the IgE molecules bonded to the surface of mast cells insensitised individuals. If two IgE molecules are crosslinked to oneanother by an allergen, this results in the release of mediators (forexample histamine, prostaglandins) and cytokines by the effector celland thus in the corresponding clinical symptoms.

A distinction is made between major and minor allergens depending on therelative frequency with which the individual allergen molecules reactwith the IgE antibodies of allergy sufferers.

In the case of timothy grass (Phleum pratense), Phl p 1 (Petersen etal., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesenand Löwenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al.,1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen etal., 1995, Int. Arch. Allergy Immunol. 108, 49-54). Phl p 2/3 (Doleceket al., 1993, FEBS 335 (3), 299-304), Phl p 4 (Haavik et al., 1985, Int.Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int.Arch. Allergy Immunol. 97: 287-294, Fischer et al., 1996, J. AllergyClin. Immunol. 98: 189-198) and Phl p 13 (Suck et al., 2000, Clin. Exp.Allergy 30: 324-332; Suck et al., 2000, Clin. Exp. Allergy 30:1395-1402) have hitherto been identified as major allergens.

The dominant major allergens of timothy grass (Phleum pratense) are Phlp 1 and Phl p 5, with Phl p 5 occurring in two forms 5a and 5b whichdiffer in respect of their molecular weight and are encoded byindependent genes. The deduced amino acid sequences both of Phl p 5a andalso of Phl p 5b have been determined by means of the recombinant DNAtechnique. Phl p 5a is a protein of about 32 kDa and reacts with the IgEantibodies of 85-90% of grass pollen allergy sufferers. Phl p 5a existsin a series of homologous variants which differ from one another throughpoint mutations and probably correspond to different allelic forms. Thepollen of related grass species, such as, for example, Lolium perenne,Poa pratensis inter alia, contains allergens which are homologous withthat of Phl p 5a and together are known as group 5 allergens. The highstructural homology of these group 5 allergens of grass species causescorrespondingly high cross reactivity of the molecules with the IgEantibodies of grass pollen allergy sufferers.

A classical approach to effective therapeutic treatment of allergies isspecific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4(6): 336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4):558-562). In this method, the patient is injected subcutaneously withnatural allergen extracts in increasing doses. However, there is a riskin this method of allergic reactions or even anaphylactic shock. Inorder to minimise these risks, innovative preparations in the form ofallergoids are employed. These are chemically modified allergen extractswhich have significantly reduced IgE reactivity, but identical T-cellreactivity compared with the untreated extract (Fiebig, 1995, Allergo J.4 (7): 377-382).

Even more substantial therapy optimisation would be possible withallergens prepared by recombinant methods. Defined cocktails ofhigh-purity allergens prepared by recombinant methods, optionallymatched to the individual sensitisation patterns of the patients, couldreplace extracts from natural allergen sources since these, in additionto the various allergens, contain a relatively large number ofimmunogenic, but non-allergenic secondary proteins.

Realistic perspectives which may result in reliable hyposensitisationwith recombinant expression products are offered by specifically mutatedrecombinant allergens in which IgE epitopes are specifically deletedwithout impairing the T-cell epitopes which are essential for therapy(Schramm et al., 1999, J. Immunol. 162: 2406-2414).

A further possibility for therapeutic influencing of the disturbed Thelper cell equilibrium in allergy sufferers is treatment withexpressible DNA which encodes for the relevant allergens(immunotherapeutic DNA vaccination). Initial experimental evidence ofallergen-specific influencing of the immune response by a DNA vaccine ofthis type has been furnished in rodents by injection ofallergen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5):540-544).

The object on which the present invention is based consisted in theprovision of novel variants of the group 5 allergens of the Pooideae atthe protein and DNA level which are distinguished by reduced IgEactivity at the same time as substantial retention of the T-cellreactivity and are therefore suitable for specific immunotherapy andimmunotherapeutic DNA vaccination.

FIGURES

FIG. 1: Alignment of relevant regions of Phl p 5a-homologous cDNAsequences of Pooideae species: Lolium perenne (Lol p), Poa pratensis(Poa p) Triticum aestivum (Tri a) and Hordeum vulgare (Hor v)

Numbering: nucleotide positions of the DNA insertions

Phl p 5a (Nucleotides 262-636 of SEQ ID NO: 1), Poa p 5 (SEQ ID NO: 15)and Lol p 5 (SEQ ID NO: 16) sequences: cDNA sequences from “GenBank”database of the National Center for Biotechnology Information (NCBI),Bethesda, USA

Hon v (SEQ ID NO: 17) and Tri a (SEQ ID NO: 18) sequences: EST sequencesfrom EST database of the Institute for Genomic Research (TIGR),Rockville, USA

Black borders: sequence identity with Phl p 5a (based on GenBankAJ555152)

Dotted borders: deletion corresponding to amino acids 94-113 (based onGenBank AJ555152)

Dashed borders: deletion corresponding to amino acids 175-198 (based onGenBank AJ555152)

FIG. 2: Alignment of Phl p 5a-homologous amino acid sequences (relevantsequence regions, deduced from DNA sequences) of Pooideae species:Lolium perenne (Lol p), Poa pratensis (Poa p) Triticum aestivum (Tri a)and Hordeum vulgare (Hor v)

Numbering: nucleotide positions of the DNA insertions

Phl p 5a (Residues 57-262 of SEQ ID NO: 21, Poa p 5 (SEQ ID NO: 19) andLol p 5 (SEQ ID NO: 201 sequences: cDNA sequences from “GenBank”database of the National Center for Biotechnology Information (NCBI),Bethesda, USA

Hor v (SEQ ID NO: 21) and Tri a (SEQ ID NO: 22) sequences: EST sequencesfrom EST database of the Institute for Genomic Research (TIGR),Rockville, USA

Black borders: sequence identity with Phl p 5a (based on GenBankAJ555152)

Dotted borders: deletion corresponding to amino acids 94-113 (based onGenBank AJ555152)

Dashed borders: deletion corresponding to amino acids 175-198 (based onGenBank AJ555152)

FIG. 3: SDS-PAGE of purified deletion mutants in the form of histidinefusion proteins

1) Marker

2) rPhl p 5a wt (His)

3) Phl p 5a DM-Δ94-113 (His)

4) Phl p 5a DM-Δ94-113, 175-198 (His)

5) Phl p 5a DM-Δ75-198 (His)

6) Marker

FIG. 4: SDS-PAGE of the purified non-fusion proteins Phl p 5aDM-D94-113, 175-198 and rPhl p 5a wt (top) and identity test with αPhl p5 antibodies (bottom)

αPhl p 5 mAb Apha-1D11 binds region 175-198

(only rPhl p 5a wt is positive)

αPhl p 5a mAb Apha-3B2 binds a joint epitope of the two Phl p 5amolecules (both proteins positive)

(mAb: monoclonal antibody)

FIG. 5: Analytical SEC of deletion mutant Phl p 5a DM-Δ94-113, 175-198and of recombinant wild type Phl p 5a (purified non-fusion proteins)

Column: Superdex 75 HR10/30 (Amersham Biosciences, Uppsala, Sweden)

Eluent: PBS

Arrow: exclusion volume

FIG. 6: Non-denaturing isoelectric focusing of deletion mutant Phl p 5aDM-Δ94-113, 175-198 and of recombinant wild type Phl p 5a (purifiednon-fusion proteins)

1) IEF marker

2) rPhl p 5a wt

3) Phl p 5a DM-Δ94-113, 175-198

pl rPhl p 5a wt=8.7

pl rPhl p 5a DM-Δ94-113, 175-198=6.4

FIG. 7: Strip test for checking the IgE binding ability of Phl p 5adeletion mutants (non-denaturing)

P: sera of clinically defined grass pollen allergy sufferers

FIG. 8: Determination of the reduced IgE reactivity of Phl p 5a deletionmutants by means of the EAST inhibition test with two representativesingle sera (a and b) and a serum pool (c)

nPh p 5a/b

rPhl p 5a wt

rPhl p 5a wt (His)

Phl p 5a DM-Δ94-113 (His)

Phl p 5a DM-Δ175-198 (His)

Phl p 5a DM-Δ94-113, 175-198

Phl p 5a DM-Δ94-113, 175-198 (His)

P: sera of clinically defined grass pollen allergy sufferers

FIG. 9: Determination of the hypoallergeneity of Phl p 5a deletionmutant Phl p 5a DM-Δ94-113, 175-198 by means of the basophil activationtest with basophils of six different grass pollen allergy sufferers (P)

DETAILED DESCRIPTION OF THE INVENTION

Mutagenesis and Cloning of cDNA Sequences

The starting point for the—particularly preferred in accordance with theinvention—hypoallergenic Phl p 5a variants is the cDNA of an isoform ofwild-type Phl p 5a which has been isolated with the aid of specificprimers by polymerase chain reaction (PCR) from the total cDNA of pollenof timothy grass (Phleum pratense) (NCBI (National Center forBiotechnology Information, Bethesda, USA) GenBank number AJ555152) (SEQID NO 1). The amino acid sequence as per SEQ ID NO 2 has been deducedfrom the cDNA sequence. Phl p 5a, which consists of 284 amino acids, wasexpressed cytosolically as soluble protein in E. coli and subsequentlypurified. This recombinant wild-type form of Phl p 5a (rPhl p 5a wt)reacts with monoclonal anti-Phl p 5 antibodies and with IgE antibodiesof grass pollen allergy sufferers which have reactivity with naturalpurified Phl p 5a (nPhl p 5a).

Starting from the described cDNA of rPhl p 5a wt, a series of differentdeletion variants (deletion mutants) was prepared byrestriction/ligation methods and PCR and ligated into the expressionvector pProExHTa (Invitrogen, Carlsbad, USA). Sections with a length of6 to 72 by distributed over the entire sequence of the cDNA moleculewere deleted, causing induction of corresponding deletions in thepolypeptide chains of the proteins expressed in E. coli.

The deletion variants of Phl p 5a were investigated by immunoblot fortheir binding ability to IgE antibodies of a representative serum poolof grass pollen allergy sufferers.

In this method, surprisingly, two deletion variants of Phl p 5a (Phl p5a DM-Δ94-113, deletion of amino acids 94-113 and Phl p 5a DM-Δ175-198,deletion of amino acids 175-198 of rPhl p 5a wt) were found, which havereduced binding of IgE antibodies (representative serum pool). These twoPhl p 5a deletions served as starting point for the construction of adouble deletion mutant containing both effective deletions (Phl p 5aDM-Δ94-113, 175-198).

The construction of Phl p 5a DM-Δ94-113, Phl p 5a DM-Δ175-198 and Phl p5a DM-Δ94-113, 175-198 by genetic engineering methods and thebiochemical and immunological characterisation thereof are describedbelow.

For the construction of deletion variant Phl p 5a DM-Δ94-113 (SEQ ID NO3, cDNA sequence (795 bp), and SEQ ID NO 4, amino acid sequence (264aa)), firstly two fragments were prepared starting from the cDNA of rPhlp 5a wt. Fragment “F1-93”, encoding for amino acids 1-93 of rPhl p 5awt, was prepared by PCR with the aid of primers 1 and 5, and fragment“F114-284” was prepared with the aid of primers 4 and 6 (primersequences see Table 1). Fragments “F1-93” and “F114-284” were employedas matrix in a further PCR using primers 1 and 4, which resulted inamplification of the complete cDNA encoding for deletion variant Phl p5a DM-Δ94-113. The basis of the connection of fragments “F1-93” and“F114-284” by PCR was a sequence region common to both fragments. Thissequence region was formed by amplification of fragment “F114-284” byPCR by means of a particular sense oligonucleotide which contained anadditional DNA sequence encoding for amino acids 88-93 in the 5′ region(Table 1).

The cDNA sequence encoding for deletion variant Phl p 5a DM-Δ175-198(SEQ ID NO 5, cDNA sequence (783 bp), and SEQ ID NO 6, amino acidsequence (260 aa)) was generated by restriction and subsequent ligationof two separately prepared cDNA fragments. The 5′-terminal fragment“F1-174” was prepared by PCR with the aid of primers 1 and 2 and the3′-terminal fragment “F199-284” with the aid of primers 3 and 4. ThecDNA fragments were digested with the restriction enzyme SpeI andsubsequently ligated (see Table 1). The ligation product was amplifiedby PCR using primers 1 and 4.

The cDNA of deletion variant Phl p 5a DM-Δ94-113, 175-198 (SEQ ID NO 7,cDNA sequence (723 bp), and SEQ ID NO 8, amino acid sequence (240 aa))was likewise prepared from two cDNA fragments. The 5′-terminal fragmentwas generated using primers 1 and 5 and with rPhl p 5a wt-cDNA asmatrix, and the 3′-terminal fragment was generated using primers 4 and 6with Phl p 5a DM-Δ175-198-cDNA as matrix. By means of the commonsequence region corresponding to amino acids 88-93 of the rPhl p 5a wtprotein, the fragments were connected by a third PCR using primers 1 and4, and the product was amplified.

The cDNAs encoding for the modified allergens were ligated into theexpression vector pProExHT (Invitrogen, Carlsbad, USA) via the EheI andHindIII restriction sites and subsequently sequenced in full.

The immunological cross reactivity of the group 5 allergens of thePooideae, such as, for example, Poa pratensis and Lolium perenne, isbased on a very similar amino acid sequence. It can be taken as certainthat the corresponding genes go back to a common progenitor gene.Homologous sequence regions in the group 5 allergens of the Pooideaeexist both for the sequences of deletions Δ94-113 and Δ175-198 of thePhl p 5a wt protein sequence (reference: GenBank AJ555152) and also forthe flanking sequence regions thereof. The high homology of the sequenceregions in question can be demonstrated both at the DNA level and alsoat the amino acid sequence level (FIG. 1 and FIG. 2).

TABLE 1 List of the PCR primers employed for the preparation of deletionvariants SEQ ID Primer NO Direction Sequence (5′→3′) 1  9 sense gcc gatcta ggc tac ggc ccg gcc 2 10 antisense aac ata act agt ggc agc gac cttgaa ggc ggc gtc 3 11 sense atc ta act agt acg ggc ggc gcc tac gaga 4 12antisense aac ata aag ctt tca gac ttt gta gcc acc agt 5 13 antisense ggagct gga ttc ggc ggc gcc ctt ggg 6 14 sense gcc gcc gaa tcc agc tcc ggcgcg acg cct gag gcc aag tac gac The SpeI restriction sites are indicatedby underliningExpression and Purification of Recombinant Phl p 5a Molecules

The recombinant proteins were expressed as histidine fusion proteinswith integrated protease cleavage site (expression vector pProExHT;Invitrogen, Carlsbad, USA) for optional removal of the histidine fusioncomponent (His) in Escherichia coli (strain JM109). rPhl p5a wt and thedeletion mutants were firstly purified by specific binding of theN-terminal histidine residues to an Ni2+ chelate matrix (immobilisedmetal ion affinity chromatography, IMAC) and subsequently by preparativegel filtration (size exclusion chromatography, SEC).

The purity of the eluted proteins was monitored by SDS-PAGE andanalytical SEC. The results showed that rPhl p 5a wt (His), Phl p 5aDM-Δ94-113 (His); Phl p 5a DM-Δ175-198 (His) and Phl p 5a DM-Δ94-113,175-198 (His) could be prepared with high purity and in monomeric form(FIG. 3). The identity of the proteins was demonstrated by Phl p5a-specific monoclonal antibodies.

The checking of the IgE reactivity by means of IgE binding techniques(immunoblotting, strip test, EAST inhibition test and basophilactivation test) and the investigation of the T-cell reactivity was inaddition carried out with test substances without a histidine fusioncomponent.

To this end, the deletion variants was prepared in parallel to thecomparative protein rPhl p 5a-wt firstly as fusion proteins. However,the histidine fusion component was subsequently cleaved offenzymatically (TEV protease, Invitrogen, Carlsbad, USA), leaving only aglycine as residue of the protease cleavage sequence on the N terminalof the target protein. Both the cleaved-off histidine component and alsothe protease used for the cleavage were separated off completely byIMAC. After preparative SEC, the purity and conformation of the elutedproteins was checked by SDS-PAGE and analytical SEC, as shown in FIGS. 4and 5 for rPhl p 5a wt and the mutant Phl p 5a DM-Δ94-113, 175-198respectively. All proteins were prepared in pure and monomeric form. Aninvestigation by non-denaturing isoelectric focusing (IEF) of thenon-fusion proteins always showed high homogeneity with respect to thesurface charge (see FIG. 6, illustrative for Phl p 5a DM-Δ94-113,175-198).

The identity of the recombinant proteins was demonstrated by themonoclonal anti-Phl p 5 antibodies (Allergopharma, Reinbek, Germany)Apha-1D11 or Apha-3B2 (see FIG. 4, illustrative for Phl p 5a DM-Δ94-113,175-198) and N-terminal sequencing.

Determination of Reduced IgE Binding of the Phl p 5a Deletion Variants

A simple test method for determination of the IgE reactivity ofallergenic molecules is investigation of the binding of specific IgEfrom the sera from allergy sufferers to membrane-bound test proteins bythe strip test.

For this purpose, the test substances are bound in the sameconcentration and amount alongside one another to a strip ofnitrocellulose membrane under non-denaturing conditions. A series ofsuch membrane strips can be incubated in parallel with various sera fromallergy sufferers. After a washing step, the specifically bound IgEantibodies are rendered visible on the membrane by a colour reactionpromoted by an anti-hIgE/alkaline phosphatase conjugate.

The IgE reactivity of the recombinant proteins Phl p 5a wt (His), Phl p5a DM-Δ94-113 (His), Phl p 5a DM-Δ175-198 (His) and Phl p 5a DM-Δ94-113,175-198 (His) was investigated comparatively in the strip test using 43individual sera from grass pollen allergy sufferers (FIG. 7).

All 43 sera from allergy sufferers contained Phl p 5a-specific IgEantibodies which reacted strongly with the natural Phl p 5a (nPhl p 5a,not shown here) and the recombinant equivalent rPhl p 5a wt (His).

Surprisingly, it became clear that the Phl p 5a-specific IgE antibodiesof all 43 patient sera did not bind at all to deletion variant Phl p 5aDM-Δ94-113, 175-198 (His) or only did so to a very greatly reducedextent. The reduced IgE binding is attributable both to the deletionΔ94-113 and also to the deletion Δ175-198. Deletion variant Phl p 5aDM-Δ175-198 (His) shows a clearly recognisably reduced IgE bindingcapacity in this test in 35 of 43 sera from allergy sufferers. In sometests, the influence of the deletion of amino acids 175-198 was so greatthat IgE binding was virtually completely prevented (Ex.: P3, P20, P28)

The influence of deletion Δ94-113 on the IgE binding reactivity is lesspronounced, but likewise clearly visible. Deletion variant Phl p 5aDM-Δ94-113 (His) was bound significantly more weakly by IgE of 19 of the43 individual sera from allergy sufferers than the reference rPhl p 5awt (His) (Ex.: P31, P37, P42). However, the reduction in the IgE bindingwas less drastically pronounced in many individual tests than thereduction caused by Δ175-198.

It is thus clear that both deletions contribute to the reduction in thetotal IgE binding reactivity of the deletion mutant Phl p 5a DM-Δ94-113,175-198 (His).

In contrast to the strip test, the EAST inhibition test (enzymeallergosorbent test) allows the investigation of allergen/IgEinteractions in solution, enabling interfering masking of epitopes ofthe test substance by immobilisation on the membrane to be fundamentallyexcluded. The EAST inhibition test is carried out as follows. Microtitreplates are coated with allergens, here natural Phl p 5 (nPhl p 5a/b,mixture of Phl p 5a and Phl p 5b). After removal of the unbound allergenmolecules by washing, the plate is blocked with bovine serum albumin inorder to prevent later non-specific binding. IgE antibodies of allergysufferers, as representative pool of individual sera (serum pool) or assingle serum, is incubated in suitable dilution with the allergen-coatedmicrotitre plates. The amount of allergen-bound IgE antibodies isquantified photometrically via an enzyme coupled to a second antibody(anti-hIgE/alkaline phosphatase conjugate) through conversion of asubstrate into a coloured end product.

The binding of the IgE antibodies is inhibited substance-specifically bya soluble allergen or the substance to be tested (recombinant modifiedallergen) depending on the concentration. Immunochemically identicalsubstances show identical inhibition curves.

The reference molecules used in this work were nPhl p 5, rPhl p 5a wt,and the histidine fusion protein rPhl p 5a wt (His). Besides othermolecules, the IgE binding of the histidine fusion proteins Phl p 5aDM-Δ94-113 (His), Phl p 5a DM-Δ175-198 (His) and Phl p 5a DM-Δ94-113,175-198 (His) and that of the non-fusion protein Phl p 5a DM-Δ94-113,175-198 was investigated by comparison with these references.

FIGS. 8 a-c show representatively the specific inhibition curves of testsubstances raised with two individual sera and a serum pool of grasspollen allergy sufferers. nPhl p 5a/b showed the greatest inhibitoryeffect in all tests (about 80-95% inhibitory effect at a concentrationof 10 μg/ml). The inhibitory effect of rPhl p 5a was significantly lowerwith a maximum inhibition of 70-80%. This effect is caused by thecomposition of nPhl p 5a/b, which also contains the isoform Phl p 5b inaddition to the isoform Phl p 5a. The specific IgE antibodies againstPhl p 5b cannot be inhibited by rPhl p 5a wt.

The histidine fusion component showed no effect on IgE binding. This isclear in all tests through the identical inhibition curves of rPhl p 5awt (His) and rPhl p 5a wt. This demonstrates the validity of tests withhistidine fusion proteins.

In general, two groups of patient sera were distinguished with respectto qualitative IgE binding.

The first group is represented by individual serum P15 (FIG. 8 a). Thesesera from allergy sufferers contained IgE antibodies whose binding toPhl p 5a was reduced by both deletions, Δ94-113 and Δ175-198. Deletionmutant Phl p 5a DM-Δ94-113 (His) showed only a maximum inhibitory effectof about 50% here, and the deletion mutant Phl p 5a DM-175-198 (His)showed an inhibitory effect of only 20-30%.

The double deletion mutant Phl p 5a DM-Δ94-113, 175-198 (His) was onlyable to inhibit the binding of IgE antibodies by 0-10% at the highestconcentration employed. The use of the non-fusion protein Phl p 5aDM-Δ94-113, 175-198 confirmed this result (0-10% maximum IgEinhibition).

The second group of sera from allergy sufferers, represented byindividual serum P44 (FIG. 8 b), differed from the first group throughthe fact that the IgE antibodies present in the sera reacted equallywell with Phl p 5a DM-Δ94-113 (His) as with the reference rPhl p 5a wt(His) (70-80% maximum inhibition), whereas no or non-detectable amountsof IgE antibodies reacted with Phl p 5a DM-Δ175-198 (His) (0-10% maximuminhibition).

The double deletion mutant Phl p 5a DM-Δ94-113, 175-198 likewise showeda greatly reduced inhibitory effect (0-10%) with this group of sera fromallergy sufferers, which was shown both for the fusion protein and alsofor the fusion component-free protein.

The sera of these allergy sufferers apparently contained IgE antibodiesdirected principally against epitopes of the C-terminal part of themolecule.

The measurement data of the IgE binding reactivity of IgE antibodies ofa serum pool of 20 allergy sufferers underline the importance of thedeletions Δ94-113 and Δ175-198 for the reduction in the IgE binding ofPhl p 5a (FIG. 8 c). Both individual deletion mutants, Phl p 5aDM-Δ94-113 (His) and Phl p 5a DM-Δ175-198 (His) show a lower maximuminhibitory effect, of 40-50% and about 30% respectively, than rPhl p 5awt (about 70%). The double deletion mutant Phl p 5a DM-Δ94-113, 175-198was only bound very weakly by the IgE antibodies of the serum pool(10-15% maximum inhibition), which, in agreement with the test of 43allergy sufferers in the strip test, indicates greatly reduced IgEbinding reactivity of this Phl p 5a variant in very many, if not all,grass pollen allergy sufferers.

Determination of the Hypoallergeneity of the Deletion Mutants byBasophil Activation Test

By means of a basophil activation test, the effects of reduced IgEbinding ability of the deletion mutants on the functional effect in thecrosslinking of membrane-bound IgE of the effector cells and activationthereof were investigated. The functional reduction in allergeneity wasthus measured in a sensitive in-vitro test.

For the basophil activation test, heparinised full blood from grasspollen allergy sufferers is incubated with various concentrations of thetest substances. Allergenic substances are able to bind specific IgEantibodies, which are associated with the high-affinity IgE receptors ofthe basophilic granulocytes.

Crosslinking of the IgE/receptor complexes initiated by the allergenmolecules results in signal transduction, which results in degranulationof the effector cells and thus initiation of the allergic reactions invivo.

In vitro, allergen-induced activation of basophilic immunocytes can bedetermined by quantification of the expression of a surface protein(CD203c) coupled to signal transduction of the IgE receptor crosslinking(Kahlert et al., Clinical Immunology and Allergy in Medicine Proceedingsof the EAACI 2002 (2003) Naples, Italy 739-744). The number of expressedsurface proteins on a cell and the percentage of activated cells of acell pool is measured highly sensitively via the binding of afluorescence-labelled monoclonal antibody to the surface protein andsubsequent analysis by fluorescence-activated flow cytometry.

The reference substances employed here were both purified natural Phl p5a (nPhl p 5a) and also rPhl p5a wt in parallel with the testsubstances. The test results of the double deletion mutant Phl p 5a DMΔ94-113, 175-198 with basophils from six test persons are shown ascurves in FIG. 9. The test results with basophils from a total of 10clinically defined allergy sufferers are shown in Table 2.

The A50 values (A50: allergen concentration at 50% of the number ofbasophils activated to the maximum) of the reference molecules were,varying individually, between ˜1.3-15 pM for rPhl p 5a wt and ˜0.3-10 pMfor nPhl p 5a (Table 2). By contrast, the A50 values of deletion variantPhl p 5a DM Δ94-113, 175-198 were between ˜18-8400 pM.

The A50 values determined for the three substances employed were used todetermine the allergenic efficacy of deletion variant Phl p 5a DMΔ94-113, 175-198 in relation to the unchanged reference molecules nPhl p5a and rPhl p5a wt for each test person (Table 2).

The relative allergenic efficacy (Pr, relative potency) of deletionvariant Phl p 5a DM Δ94-113, 175-198 was reduced between ˜12-5000 foldcompared with the reference rPhl p 5a wt or ˜16-32000 fold compared withthe reference nPhl p 5a (Table 2).

TABLE 2 Determination of the hypoallergeneity of deletion mutant Phl p5a DM-Δ94-113, 175-198 by means of basophil activation test Pr value^(b)Pr value^(b) Phl p 5a Phl p 5a Test substance DM-Δ94- DM-Δ94- A₅₀[pM]^(a) 113, 113, Phl p 5a 175-198 175-198 nPhl p rPhl p 5a DM-Δ94-113,relative to relative to Donor^(c) 5a wt 175-198 rPhl p 5a wt^(d) nPhl p5a^(e) P13 4.08 5.34 477.2 0.0111 0.0085 P17 6.44 2.68 466.6 0.00570.0137 P20 0.26 1.68 8433.0 0.0002 ^(f) 0.00003 ^(f) P23 1.02 1.26 39.20.0321 0.0260 P24 1.22 2.57 58.1 0.0442 0.0209 P28 9.43 11.35 198.20.0573 0.0476 P29 1.77 2.34 33.7 0.0694 0.0525 P31 10.15 14.66 3967.00.0037 0.0026 P34 3.48 2.54 165.1 0.0153 0.0211 P40 1.08 1.45 17.50.0829 0.0617 ^(a)Allergen concentration at 50% of the number ofbasophils activated to the maximum ^(b)Relative potency ^(c)Clinicallydefined grass pollen allergy sufferers ^(d)Calculated from A50 rPhl p 5awt/A50 Phl p 5a DM-Δ94-113, 175-198 ^(e)Calculated from A50 nPhl p5a/A50 Phl p 5a DM-Δ94-113, 175-198 ^(f)Bold: minimum and maximum valuesT-Cell Reactivity

T helper lymphocytes react with peptide fragments of the allergens(approx. 12-25 amino acids) formed by enzymatic degradation inantigen-presenting cells (APCs) and are presented to the T-cells afterinclusion of the suitable peptides in the individual MHC class IImolecules at the surface of the APCs. This allergen-specific activationof the T helper lymphocytes is the prerequisite for subsequent reactions(proliferation, anergy, apoptosis) and for functional differentiation(TH1 and TH2). The influencing of allergen-specific T-lymphocytes bytreatment with an allergen or an allergen variant in hyposensitisationis regarded as the key for the therapeutic efficacy.

In order to investigate T-cell reactivity, oligoclonal T-cell lines(TCLs) of Graminae pollen allergy sufferers are established byconventional methods with stimulation by nPhl p5 or rPhl p 5 molecules.

In a proliferation test, the various T-cell lines were stimulated withthe reference allergens nPhl p5a and rPhl p5a wt and the double deletionmutant Phl p 5a DM Δ94-113, 175-198. The proliferation rate wasdetermined by the incorporation of [³H] thymidine by conventionalmethods.

TABLE 3 Determination of the T-cell reactivity of deletion mutant Phl p5a DM-Δ94-113, 175-198 by means of proliferation tests with Phl p5-specific T-cell lines (TCLs) Stimulation index^(a) Phl p 5a rPhlDM-Δ94-113, Donor^(b) TCL nPhl p 5a p 5a wt 175-198 A 3.2 9.8 4.9 4.4 B8.2 21.0 15.5 13.3 C 11.2 5.2 4.7 7.2 C 11.3 3.3 2.9 3.5 C 11.43 3.0 3.92.6 D 19.1 6.5 4.7 7.5 D 19.2 9.6 3.3 2.6 E 23.22 21.8 29.0 20.8 E 23.507.5 8.4 6.6 F 89.23 1.8 3.5 1.8 ^(a)Calculated from [³H] measurementvalues. cpm measurement values of allergen-stimulated cell cultures/cpmmeasurement values of unstimulated cell cultures ^(b)Donor: clinicallydefined grass pollen allergy sufferers

The results with ten TCLs from six allergy sufferers show that theseTCLs were stimulated to proliferation by Phl p 5a DM Δ94-113, 175-198 incomparable strength as by the unchanged natural or recombinant wild-typeallergen (Table 3).

The present invention thus relates to variants of the group 5 allergensof the Pooideae which are characterised by reduced IgE reactivitycompared with the known wild-type allergens and by retained reactivitywith T-lymphocytes. These group 5 allergens are preferably Phl p 5a, Poap 5 and Lol p 5, very particularly preferably Phl p 5a.

As it has proven particularly favourable for the purposes of theinvention for amino-acid sequence regions which correspond to amino-acidsequence regions 94-113 and 175-198 of Phl p 5a to be missing or removedin the group 5 allergens, this invention relates, in particular, to suchallergen variants. The first-mentioned or second-mentioned region may bemissing individually, but also both said regions may be missingsimultaneously, with the latter embodiment being very particularlypreferred.

Owing to the high sequence homologies within the group 5 allergens fromPooideae, these regions can be unambiguously identified in sequencealignments of the Phl p 5a sequence with sequences from other group 5allergens. The above-described allergen variants preferably originatefrom Phl p 5a or correspond to the sequences in accordance with SEQ IDNO 4, 6 or 8.

The allergen variants according to the invention can be preparedstarting from the cloned DNA sequence with the aid of geneticengineering methods. In principle, however, chemical modifications ofthe native allergen extract are also possible (Fiebig, 1995, Allergo J.4 (7), 377-382).

Naturally, further modifications in other positions—for example in orderto increase the hypoallergeneity—are also possible via the variations ofgroup 5 allergens described in the present patent application. Thesemodifications can be, for example, amino acid insertions, deletions andexchanges, cleavage of the protein into fragments and fusion of theprotein or fragments thereof with other proteins or peptides.

During preparation of the allergen variants described in more detailhere, an His tag was introduced by genetic engineering methods for thepurposes of improved purification of the overexpressed proteins.

The invention furthermore relates to a DNA molecule encoding for anallergen variant described above, in particular corresponding to asequence in accordance with SEQ ID NO 3, 5 or 7, to a recombinantexpression vector containing this DNA molecule, and to a host organismtransformed with said DNA molecule or said expression vector. Suitablehost organisms may be prokaryotic or eukaryotic, single- or multicelledorganisms, such as bacteria or yeasts. A host organism which ispreferred in accordance with the invention is E. coli.

The invention furthermore relates to a process for the preparation of anallergen variant according to the invention by cultivation of the saidhost organism and isolation of the corresponding allergen variant fromthe culture.

The present invention additionally relates to the allergen variants, DNAmolecules and expression vectors described above in their property asmedicaments.

The present invention furthermore relates to pharmaceutical compositionscomprising at least one of these allergen variants or a correspondingDNA molecule or a corresponding expression vector and optionally furtheractive ingredients and/or adjuvants for the treatment of allergies inthe triggering of which group 5 allergens of the Pooideae are involved,or for the immunotherapeutic vaccination of patients having allergies inthe triggering of which group 5 allergens of the Pooideae are involvedand/or for the prevention of such allergies.

If these are pharmaceutical compositions of the second type (comprisingat least one DNA molecule or an expression vector), these compositionspreferably furthermore comprise aluminum hydroxide, an immunostimulatoryCpG-containing oligonucleotide or a combination of the two as adjuvants.

For the purposes of this invention, pharmaceutical compositions can beused as therapeutic agents in human or veterinary medicine. Suitableexcipients are organic or inorganic substances which are suitable forparenteral administration and do not react with group 5 allergenvariants according to the invention. Suitable for parenteraladministration are, in particular, solutions, preferably oily or aqueoussolutions, furthermore suspensions, emulsions or implants. The allergenvariants according to the invention may also be lyophilised and theresultant lyophilisates used, for example, for the preparation ofinjection preparations. The compositions indicated may be sterilisedand/or comprise adjuvants, such as lubricants, preservatives,stabilisers and/or wetting agents, emulsifiers, salts for modifying theosmotic pressure, buffer substances and/or a plurality of further activeingredients.

Furthermore, appropriate formulation of the allergen variants accordingto the invention enables depot preparations to be obtained, for exampleby adsorption on aluminum hydroxide.

Finally, the present invention relates to the use of at least oneallergen variant according to the invention or a DNA molecule accordingto the invention or an expression vector according to the invention forthe preparation of a medicament for the treatment of allergies in thetriggering of which group 5 allergens of the Pooideae are involved orfor the immunotherapeutic vaccination of patients having allergies inthe triggering of which group 5 allergens of the Pooideae are involvedand/or for the prevention of such allergies.

We claim:
 1. A polypeptide which comprises a polypeptide sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6 and SEQID NO:
 8. 2. The polypeptide according to claim 1, which comprises thepolypeptide sequence of SEQ ID NO:
 4. 3. The polypeptide according toclaim 1, which is obtained by recombinant genetic engineering methods.4. A process for the preparation of at least one polypeptide accordingto claim 1 comprising culturing a host organism transformed with a DNAencoding said at least one polypeptide according to claim 1 or a vectorcomprising said DNA encoding said at least one polypeptide according toclaim 1; and isolating the polypeptide from the culture.
 5. A medicamentcomprising at least one polypeptide according to claim 1 and anexcipient.
 6. A pharmaceutical composition comprising at least onepolypeptide according to claim 1 and a further active ingredient oradjuvant.
 7. A method for treating an allergy triggered by a group 5allergen of Pooideae species, comprising administering to a subject inneed thereof at least one polypeptide according to claim
 1. 8. Thepharmaceutical composition according to claim 6, wherein the adjuvant isaluminum hydroxide, an immunostimulatory CpG-containing oligonucleotideor a combination thereof.
 9. A method for the treatment of an allergytriggered by group 5 allergens of Pooideae species, comprisingadministering to a subject in need thereof the pharmaceuticalcomposition according to claim
 6. 10. A variant polypeptide whichconsists of (a) amino acids 1-37 and 58-206 of SEQ ID NO: 19; (b) aminoacids 1-118 and 143-206 of SEQ ID NO: 19; or (c) amino acids 1-37,58-118 and 143-206 of SEQ ID NO:
 19. 11. A variant polypeptide whichconsists of (a) amino acids 1-33 and 54-204 of SEQ ID NO: 20; (b) aminoacids 1-116 and 141-204 of SEQ ID NO: 20; or (c) amino acids 1-33,54-116 and 141-204 of SEQ ID NO:
 20. 12. A variant polypeptide whichconsists of (a) amino acids 1-39 and 60-208 of SEQ ID NO: 21; (b) aminoacids 1-120 and 145-208 of SEQ ID NO: 21; or (c) amino acids 1-39,60-120 and 145-208 of SEQ ID NO:
 21. 13. A variant polypeptide whichconsists of (a) amino acids 1-39 and 60-210 of SEQ ID NO: 22; (b) aminoacids 1-122 and 147-210 of SEQ ID NO: 22; or (c) amino acids 1-39,60-210 and 147-210 of SEQ ID NO:
 22. 14. A polypeptide according toclaim 1, which is an isolated polypeptide.